Pathological Characteristics of Triple-Negative Breast Cancer at Main Referral Teaching Hospital, April 2014 to April 2015, Tehran, Iran.

Background: Triple-negative breast cancers (TNBC) are defined as breast cancers with lack of estrogen and progesterone receptors and no overexpression of human epidermal growth factor receptor 2 (HER2). This study was performed to determine the frequency and pathologic features of TNBC in Iranian patients. Subjects and Methods: This cross-sectional study was performed on patients with breast cancer who referred to Cancer Institute, affiliated to Tehran University of Medical Sciences, from April 2014 to April 2015. Data about the demographics, the status of gene receptors and the pathologic features were extracted from patients' records. Results: Of 214 pathology samples of patients with malignant breast cancer, TNBCs account for 14% of cases. The mean age in N-TNBC group was 50 ± 12 years. Significant difference was seen between the age of two groups (p=0.03). No significant difference was observed regarding the number of involved lymph nodes between two groups (p=0.058). Presence of vascular and nerve invasion and involvement of surgical margins at the time of diagnosis were significantly more frequent in TNBC group comparing with N-TNBC. Grade III of histologic and nuclear grading was significantly more common in TNBC. Conclusion: TNBC group was significantly associated with higher grade, higher mitotic indices and higher rate of P53 positivity and higher level of Ki-67 at the time of diagnosis. High grade breast cancers are more seen in TNBC. The presence of aforementioned characteristics in a patient highlights the need for evaluating TNBC biomarkers to better predict prognosis and consider appropriate treatment.


INTRODUCTION
Breast cancer (BC) is the second common cancer worldwide, accounts for about 10.4% of all cancers. It is also the second common cause of cancer death in females. [1][2][3] Breast cancer is the main cause of mortality in women aged 45 to 55. [3][4] In 2013, nearly 234,000 women were diagnosed with breast cancer in US, 39,000 of who have died. 5 Diagnostic evaluation of breast cancers is used commonly with immunohistochemistry (IHC) staining for three biomarkers of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor2 (HER2). 4 One of the most challenging breast cancer types is TNBC. Triple-negative breast cancer (TNBC) is referred to a type of BC which is negative for these three mentioned biomarkers. Indeed, in TNBC patients, genes of these three biomarkers are not expressed. Despite the similarity in basic diagnosis of TNBC and other types of BC,

201
International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir different factors distinguish this type of cancer from other types. Some of these factors are difference in the age of patient at time of diagnosis, race, risk factors, pathologic and molecular properties, normal course of this disease, sensitivity and response to chemotherapy. 5,6 This type of cancer often has a more aggressive nature compared with other types of breast cancer; then, routine hormonal treatments are ineffective for that. 7 In recent years, TNBC has attracted the attention of therapeutic and counseling cancer centers in different countries. 7 TNBC is an interesting subject for research due to the following five reasons: 1) TNBC is considered a bad prognostic actor for disease free survival and overall survival, 2) No effective treatment has so far been developed for this type of cancer, 3) This cancer is further seen in women of pre-menopause age and of African race, 4) There is a significant overlapping between Basal like phonotype and TNBC and 5) There is a significant overlapping between TNBC and BRCA1 enriched breast cancers. 8 TNBC is often diagnosed with a high-grade ductal histology and the increased amount of mitosis and cell proliferation. 9 Due to lack of hormone receptors and HER2 expression and subsequently, lack of response to hormone treatments and Transtuzumab, there is weak early warning sign. 9 Compared with other subtypes, metastasis to viscera particularly lung and brain and to bones is less common. 10 Furthermore, this subtype has a low survival and high relapse particularly over three to five years following diagnosis. 9 No standard treatment regimen has been registered for TNBC and also, there is insufficient information available to that affect. Although this type of cancer is initially sensitive to chemotherapy, it is significantly more invasive than other tumors. 11 Regarding the high prevalence of BC in Iranian females, geographic variation in distribution and clinic-pathological specifications of this cancer, this study aims to determine demographics and histopathologic features of this type of breast cancer (TNBC) in Iran and then, comparing that with non-TNBC (N-TNBC).

SUBJECTS AND METHODS Study population
This cross-sectional study was performed on patients with breast cancer, who referred to Cancer Institute, affiliated to Tehran University of Medical Sciences from April 2014 to April 2015. All pathology sample records of patients with breast mass which were diagnosed as a malignant breast tumor were included in this study. Samples with absence or incomplete immunohistochemistry report for the respective pathology were excluded. Immunohistochemically stained slides were evaluated for the presence of positive reaction, cellular localization (nuclear or cytoplasmic), pattern of staining (focal or diffuse) and intensity of reaction in individual tumor cells (strong or weak). Any positive nuclear reaction for ER and PR, irrespective of percentage of reactive cells, was recorded as positive. The IHC test gives a score of 0 to 3+ that measures the amount of HER2 receptor protein on surface of cells in a breast cancer tissue sample. If the score is 0 to 1+, it is called "HER2 negative" (No or weak staining). Incomplete membrane staining in any % of cells is defined as score 1+. If the score is 2+, it is called "borderline, Equivocal" (Strong complete homogeneous membrane staining (chicken wire pattern) in ≤30% of cells or weak/moderate heterogeneous complete membrane staining in at least 10% of cells). A score of 3+ is called "HER2 positive" (Strong complete homogeneous membrane staining (chicken wire pattern) in >30% of cells). Ki-67 is a nuclear non-histone protein that is present at low levels in quiescent cells but is increased in proliferating cells, especially in the G2, M and latter half of the S phase. Only nuclear staining (plus mitotic figures which are stained by Ki-67) should be incorporated into the Ki-67 score that is defined as the percentage of positively stained cells among total number of malignant cells scored. Scoring should involve the counting of at least 500 malignant invasive cells (and preferably at least 1000 cells). The ethics committee of Tehran University of Medical Sciences approved the study protocol according to the declaration of Helsinki. Malignant breast tumors with negative status for ER, PR, HER2 and IHC biomarkers were defined as TNBC group and were considered as case group.

202
International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir Other patterns which were N-TNBC group which were considered as control group. According to pathology reports, frequency, age, sex, cancer type, tumor size, tumor grade, tumor location, the benign accompanied lesion, presence of lymph node involvement and the number of involved lymph nodes, presence or absence of in situ component, skin involvement, nipple involvement, involvement of the surgical margins, vascular invasion or perineural invasion, mitoses, Ki-67 proliferative factor percent, necrosis, nuclear grade, calcification and granulomatous reaction were compared between two groups.

Data analysis
The statistical package of social science, version 19.0 (SPSS, Chicago, Illinois, USA) was used for data analysis. Statistical significance was noted for p≤0.05. For finding the association between qualitative variables, Chi-Square test was used, while independent sample t-test and ANOVA test were applied for comparison of quantitative variables.

RESULTS
Two hundred and fourteen pathology samples of patients with breast cancer were evaluated. Thirty patients (14%) were negative for all three receptors (TNBC group) and 184 patients (86%) belonged to N-TNBC group. The mean age of patients in TNBC group was 43 ± 12 years (26 to 85 years). The mean age in N-TNBC group was 50 ± 12 years (24 to 91 years). Significant difference was seen between the age of two groups (p=0.03). One hundred and eighty one of the patients (98.4%) were female and 3 patients (1.6%) were male in N-TNBC group. TNBC group were all females. Significant sex difference was not seen between two groups (p=0.48). Tumor size in the TNBC group was 3.83 ± 1.88 within the 1-10 cm range and was 2.98 ± 2.22 in N-TNBC group, within the 0.2-13 cm range. Although the mean size of tumor is greater in TNBC group, there was no significant differences regarding the tumor size between two groups (p=0.72). The number of involved lymph nodes in TNBC group was 3 ± 3 within the 0-22 range and in N-TNBC was 2 ± 2 within the 0-7 range. No significant difference was observed regarding the number of involved lymph nodes between two groups (p=0.058). TNBC group was significantly associated with younger age, higher grade, higher mitotic indices and higher rate of P53 positivity and higher level of Ki-67 at the time of diagnosis. Presence of vascular and nerve invasion and involvement of surgical margins at the time of diagnosis were significantly more frequent in TNBC group comparing with N-TNBC. Table 1 shows other pathologic specifications of breast tumor in these two groups. Grade III of cellular and nuclear grading as well as involvement of left lower quadrant (LLQ) was significantly more common in TNBC. The microscopic and macroscopic characteristics of breast tumor of two groups are compared in Table 2.

DISCUSSION
In present study, the prevalence of TNBC among Iranian patients with breast cancer found to be 14%. TNBC is associated with younger age, higher grade, higher mitotic indices, higher P53 positivity and higher level of Ki-67 at the time of diagnosis. Besides, vascular and nerve invasion and invasion to surgical margins were more frequently seen in TNBC compared with N-TNBC tumors. The in situ components of both ductal and lobular types were significantly more present in TNBC but it was true International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir poorer prognosis, if its grade is higher. So, in our study cellular and nuclear grading were both evaluated and higher cellular grading is reported in TNBC of Iranian race. Cellular grading was grade II in 82.6% of patients and the second most common was grade III (9.2%). Also, the common nuclear grading was grade II (79.9%) and the second most common was grade III (12.5%). This result indicates that high grade breast cancers are more seen in TNBC, while in N-TNBC group, grade I was in second position, in both cellular and nuclear grading.

CONCLUSION
The presence of aforementioned characteristics in patient highlights the need for evaluating TNBC biomarkers, to better predict the prognosis and consider appropriate treatment.